This application proposes to investigate the vitamin D receptor (VDR) and to examine its role in the action of 1,25-dihydroxyvitamin D3 (1,25D), the active form of vitamin D. The focus of the proposal is the study of the VDR and various other cellular factors that modulate the amplitude and nature of the target tissue responses to 1,25D. 1,25D responsiveness may be increased or decreased by a variety of cellular mechanisms, yielding a spectrum of activity in target cells from highly sensitive to totally resistant. Three Specific Aims are to be addressed in the investigation of factors that contribute to the regulation of 1,25D responsiveness. Specific Aim I will probe into the genetic mechanisms of target organ resistance due to mutations in the VDR. It will examine a number of new families with the syndrome of target organ resistance to 1,25D, Hereditary Vitamin D Resistant Rickets (HVDRR), and elucidate the nature of the mutations in the VDR gene. In addition, it will determine whether 1,25D- independent activation of target genes is the mechanism by which some HVDRR children "outgrow" their disease. Specific Aim II will investigate the role of proteins that interact with the VDR (VDR- Interacting Proteins or DRIPs) in mediating 1,25D action. Three areas in which DRIPs will be studied include: 1) a search for new and unknown DRIPs employing the GAL4 Two-Hybrid System; 2) investigation by mutational and functional analyses of various aspects of the interaction of VDR with the retinoid X receptor (RXR), with which it is known to heterodimerize to transactivate target genes; 3) exploration of the role of calreticulin, a protein recently shown to inhibit the action of other steroid receptors and that of VDR (as shown by the applicants' preliminary data). Specific Aim III will study the physiological mechanisms by which VDR abundance and function is modulated by retinoids, glucocorticoids and estrogens. Special attention will be addressed to the prostate gland and prostate cancer cells. There are many health- related aspects of the project including: for Aim I, the elucidation of mutations in HVDRR families and providing genetic counseling, as well as delineating the mechanisms by which some children with HVDRR "outgrow" their genetic defect and heal their rickets; for Aim II, investigating multiple factors that modulate 1,25 responsiveness in normal cells and potentially in various disease states; and for Aim III, exploration of the interaction of 1,25 with retinoids, estrogens and glucocorticoids, all of which have been used to treat prostate cancer, to determine whether an enhancement of the beneficial effects of 1,25D can be achieved in the therapy of prostate cancer. Overall, the grant request is a multi-faceted analysis of the role of VDR in 1,25D action in various states of health and disease, emphasizing the themes of genetic and physiological regulation of hormone responsiveness.